Large ion channels are recognized as both the gateways of metabolite exchange and multifunctional membrane receptors. They are also critical components of many toxins. To study channels under precisely controlled conditions, we purify the channel-forming proteins produced by different organisms and then reconstitute them into planar lipid bilayer membranes. We explore a number of channel-forming proteins which include mammalian VDAC (Voltage-Dependent Anionic Channel from the outer membrane of mitochondria), Anthrax Protective Antigen (from Bacillus anthracis), OmpF (general bacterial porin from Escherichia coli), LamB (sugar-specific bacterial porin from Escherichia coli), alpha-Hemolysin (toxin from Staphylococcus aureus), OprF (porin from Pseudomonas aeruginosa), Alamethicin (amphiphilic peptide toxin from Trichoderma viride), and Syringomycin E (lipopeptide toxin from Pseudomonas syringae). We think that combining work with this broad variety of channels of different origin, structure, and function in one laboratory is the most effective strategy towards elucidation of major physical mechanisms of their regulation. I. Physical theory of channel-facilitated metabolite transport. Our effort in physical theory concentrates on further development of the continuum diffusion model of solute dynamics in a membrane channel. The most important advance of this year was to apply our analytical approach to the so-called entropy potentials and to show the importance of particle-particle interactions in breaking the particle flux symmetry. Water-filled pores of biological channels usually have complex geometry that only rarely can be approximated by a cylinder. For example, high-resolution crystallography of bacterial porins and other large channels demonstrates that their pores can be envisaged as tunnels whose cross-sections change significantly along the channel axis. For some of them, variation in cross-section area exceeds an order of magnitude, which leads to the so-called entropic wells and barriers in theoretical description of transport through such structures. To approach this complex problem, we analyzed transport through conical channels that is driven by the difference in particle concentrations on the two sides of the membrane. Indeed, fluxes of non-interacting particles through the same channel, inserted into the membrane in two opposite orientations, are equal because of the detailed balance. We have shown that this flux symmetry is broken by particle-particle interactions, so that one of the orientations the orientation corresponding to the increasing channel cross section in the direction of transport can be much more efficient under the same external conditions. Our analytical results were confirmed using three-dimensional Brownian dynamics simulations. II. Physical interactions in transport regulation. This year we were able to address a wide scope of questions ranging from van der Waals interactions of particles in liquids to electrostatic effects in channel selectivity and conductance. One of our achievements was the elucidation of the lipid-packing-dependent partitioning of the prototypical anesthetic halothane into lipid bilayers. Using gramicidin A channel as a molecular probe, we found that its sensitivity to the anesthetic is highly lipid dependent. Specifically, exposure of membranes made of lamellar DOPC to halothane in concentrations close to clinically relevant reduces channel life-times by an order of magnitude. At the same time, gramicidin channels in membranes of non-lamellar DOPE are little, if at all, affected by halothane. We attribute this difference in channel behavior to a difference in the stress of lipid packing into a planar lipid bilayer, wherein the higher stress of DOPE packing reduces halothane partitioning into the hydrophobic interior. Thus, our findings suggest a new role for the physical interactions originating from the stress of lipid packing, revealing a previously unknown mechanism of anesthetic efficacy regulation. Another achievement was to use an ion channel nanopore as a single-molecule sensor to follow the effect of different salts on the adamantane-cyclodextrin complexation reaction. Since the pioneering experiments of Hofmeister on the salting out of aqueous solutions of proteins, a vast body of research has taught us that cosolutes can increase or decrease complex stability, whether preferentially excluded from or attracted to the surfaces of the associating molecules. Now studies of the underlying kinetics at a single-molecule level open the possibility of instructive inquiry into the molecular basis of the phenomenon. Surprisingly, we found that not only the stability of the complex, as measured by the life time of the inclusion complex, but also the accessibility of the complexation site can be critically increased by preferentially excluded cosolutes. Significance of these findings follows from the fact that all biochemical reactions, such as protein folding, drug binding to the target, protein-ligand and protein-DNA interactions, require release or restructuring of water layers associated with interacting molecular surfaces in the crowded environment of the cell. Therefore, understanding the mechanisms of molecular surface hydration is crucially important in resolving the fundamental questions of molecular recognition and assembly, drug design, and transport regulation. III. A new role for the old protein: Tubulin in regulation of mitochondria respiration. Regulation of mitochondrial outer membrane (MOM) permeability has dual importance: in normal metabolite and energy exchange between mitochondria and cytoplasm and thus in control of respiration, and in apoptosis by release of apoptogenic factors into the cytosol. However, the mechanism of this regulation involving the voltage-dependent anion channel (VDAC), the major channel of MOM, remains controversial. A long-standing puzzle is that in permeabilized cells, adenine nucleotide translocase (ANT) is less accessible to cytosolic ADP than in isolated mitochondria. We solve this puzzle by finding a missing player in the regulation of MOM permeability: the cytoskeletal protein tubulin. We show that nanomolar concentrations of dimeric tubulin induce voltage-sensitive reversible closure of VDAC reconstituted into planar phospholipid membranes. Tubulin strikingly increases VDAC voltage sensitivity and at physiological salt conditions could induce VDAC closure at <10 mV transmembrane potentials. Experiments with isolated mitochondria confirm these findings. Tubulin added to isolated mitochondria decreases ADP availability to ANT, partially restoring the low MOM permeability (high apparent Km for ADP) found in permeabilized cells. Our findings suggest a previously unknown mechanism of regulation of mitochondrial energetics, governed by VDAC and tubulin at the mitochondria-cytosol interface. This tubulin-VDAC interaction requires tubulin anionic C-terminal tail peptides. The significance of this interaction may be reflected in the evolutionary conservation of length and anionic charge in the C-terminal tails throughout eukaryotes, despite wide changes in the exact sequence. Additionally, tubulins that have lost significant length or anionic character of C-terminal tails are only found in cells that do not have mitochondria. To conclude, the discovered VDAC/tubulin interaction provides a previously absent link between Ca2+ homeostasis, cytoskeleton/microtubule activity, mitochondria, and apoptosis. This may help to identify the mechanisms of mitochondria-associated action of chemotherapeutic microtubule-targeting drugs such as paclitaxel and vincristine and also to understand why and how cancer cells preferentially use inefficient glycolysis rather than oxidative phosphorylation (Warburg effect).